Operator`s manual

40
LightCycler® 480 Gene Scanning Software
Performing Gene Scanning Analysis
To customize the Difference Plot display:
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In the Difference Plot tab, click either above the upper or lower chart. The
chart’s options toolbar is displayed, containing a chart menu.
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To change the chart type, select the new chart type from the respective Chart menu.
For the upper chart you have the following choices:
Melting Curves: corresponds to the ► Melting Curve display in the Negatives tab, i.e.,
it shows the unnormalized and non-temperature shifted curves
Normalized Curves: displays the normalized melting curves ►
Normalized and Temp-Shifted Curves: corresponds to the ► Normalized and Shifted
Melting Curves chart in the Temperature Shift tab.
The normalized and shifted melting curves are basis for calculating the Differ-
ence Plot.
For the lower chart you have the following choices:
Melting Peaks: displays the Melting Peaks plot (negative first derivative of the ►
sample fluorescence plotted against temperature, corresponds to the result of the
T
m
Calling Analysis Module)
Normalized and Temp-Shifted Difference Plot: displays the normalized and ►
temperature-shifted Difference Plot (see Step 8 above)
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To change the sensitivity setting:
You can adjust the Sensitivity value to refine the Gene Scanning result. The Sensitivity
function lets you influence the stringency with which melting curves are classified into dif-
ferent groups. A high Sensitivity value generally produces more groups than a low value.
Click the Sensitivity tab in the Results table to modify the analysis setting:
Slide the Sensitivity slider bar to the left to reduce stringency, or to the right to increase
stringency.
The default value is 0.3 (recommended for initial analysis). This value provides a reason-
able balance between producing too many unknowns and generating potentially wrong
calls.
Please note, that in LightCycler® 480 Gene Scanning Software version 1.5 the sensitivity
calculation has changed. This means that an analysis defined in an earlier version with a
specific sensitivity value (e.g., “0,95”) will not have the same results as an analysis defined
in version 1.5 with the same sensitivity value.